HER3 inhibitor for modulating radiosensitivity

ABSTRACT

The present invention relates to the use of an inhibitor of HER-3 for the treatment of a hyperproliferative disease in combination with radiation treatment.

This application is a Divisional of U.S. patent application Ser. No. 14/380,268, filed Aug. 21, 2014, which is the National Stage of International Patent Application No. PCT/EP2013/053562, filed Feb. 22, 2013, which claims the benefit of priority of Provisional Application No. 61/602,239, filed Feb. 23, 2012, the disclosures of which are expressly incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format, and which is hereby incorporated by reference in its entirety.

This invention was made with government support under grant UL1TR000427 awarded by the National Institutes of Health, National Center for Advancing Translational Sciences. The government has certain rights in the invention.

The present invention relates to the use of an inhibitor of HER-3 for the treatment of a hyperproliferative disease in combination with radiation treatment.

The human epidermal growth factor receptor 3 (HER-3, also known as ErbB3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER-1 (also known as EGFR), HER-2, and HER-4 (Plowman et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4905-4909; Kraus et al., Proc. Natl. Acad. Sci. U.S.A. 86 (1989), 9193-9197; and Kraus et al., Proc. Natl. Acad. Sci. U.S.A. 90 (1993), 2900-2904). Like the prototypical epidermal growth factor receptor, the transmembrane receptor HER-3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain.

The ligand Heregulin (HRG) binds to the extracellular domain of HER-3 and activates the receptor-mediated signaling pathway by promoting dimerization with other human epidermal growth factor receptor (HER) family members and transphosphorylation of its intracellular domain. Dimer formation between HER family members expands the signalling potential of HER-3 and is a means not only for signal diversification but also signal amplification. For example the HER-2/HER-3 heterodimer induces one of the most important mitogenic signals among HER family members.

HER-3 has been found to be overexpressed in several types of cancer such as breast, gastrointestinal and pancreatic cancers. Interestingly a correlation between the expression of HER-2/HER-3 and the progression from a non-invasive to an invasive stage has been shown (Alimandi et al., Oncogene 10,1813-1821; deFazio et al., Cancer 87, 487-498; Naidu et al., Br. J. Cancer 78, 1385-1390). Accordingly, agents that interfere with HER-3 mediated signaling are desirable. Murine or chimeric HER-3 antibodies have been reported, such as in U.S. Pat. Nos. 5,968,511, 5,480,968 and WO03013602.

The object of the present invention was to provide treatment of hyperproliferative diseases, in particular cancer, based on the inhibition of HER-3 being more effective than known from the prior art. This object is solved by the use of an inhibitor of HER-3 for the treatment of a hyperproliferative disease in combination with radiation treatment.

According to a first aspect the present invention relates to the use of an inhibitor of HER-3 for the treatment of a hyperproliferative disease in combination with radiation treatment.

The treatment of a combination of a HER-3 inhibitor and radiotherapy leads to synergistic effects which exceed the advantages of a treatment with either a HER-3 inhibitor or radiotherapy alone.

An inhibitor of HER-3 according to the present invention may act on the protein level or on the nucleic acid level. Examples for inhibitors acting on the nucleic acid level are known to the person skilled in the art and comprise antisense molecules, RNAi molecules and/or ribozymes.

Examples for an inhibitor acting on the protein level is an anti-HER-3-antibody or an antigen-binding fragment thereof as well as scaffold proteins. The inhibitor being an anti-HER-3 antibody or a fragment thereof, in particular an antigen-binding fragment thereof, represents a preferred embodiment of the present invention. According to the invention term “antibody fragment” comprises any portion of the afore-mentioned antibodies, preferably their antigen binding or variable regions.

As used herein, “scaffold protein” represents an protein having an antibody like binding activity or an antibody, i.e. an anti-HER-3 antibody. Within the context of the present invention, the term “scaffold protein”, as used herein, means a polypeptide or protein with exposed surface areas in which amino acid insertions, substitutions or deletions are highly tolerable. Examples of scaffold proteins that can be used in accordance with the present invention are protein A from Staphylococcus aureus, the bilin binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins, and human fibronectin (reviewed in Binz and Plückthun, Curr Opin Biotechnol, 16, 459-69). In addition, a scaffold protein having an antibody like binding activity can be derived from an acceptor polypeptide containing the scaffold domain, which can be grafted with binding domains of a donor polypeptide to confer the binding specificity of the donor polypeptide onto the scaffold domain containing the acceptor polypeptide.

The anti-HER-3 antibody according to the invention may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antigen-binding fragment thereof. The use of a monoclonal antibody is especially preferred. A person skilled in the art knows how to produce such antibodies. Monoclonal antibodies may, e.g., be produced by any suitable method such as that of Köhler and Millstein (Nature, 1975; 256:495-497).

An antibody fragment according to the invention is preferably a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fv fragment, a diabody, or a single chain antibody molecule. The “Fab fragment” differs from the “Fab′ fragment” by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. The “F(ab′)₂ fragment” originally is produced as a pair of “Fab′ fragments” which have hinge cysteines between them. In accordance with the present invention, the “Fv fragment” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site.

The antibody used according to the invention may be preferably of the IgG1, IgG2, IgG3 or IgG4 antibody type.

Humanized forms of antibodies represent a further preferred embodiment and may be generated according to the methods known in the art such as humanization or CDR grafting. Alternative methods for producing humanized antibodies are well-known in the art and described, for instance, in EP 0 239 400 and WO 90/07861. Human antibodies avoid certain of the problems associated with xenogeneic antibodies, for example antibodies that possess murine or rat variable and/or constant regions. Monoclonal humanized anti-HER-3 antibodies represent an especially preferred embodiment of the invention.

According to a further preferred embodiment the antibody is directed against the extracellular domain of HER-3. The anti-HER-3 antibody preferably interacts with at least one epitope in the extracellular part of HER-3. The epitopes are preferably located in domain L1 (AA19-184), which is the amino terminal domain, in domain S1 (AA185-327) and S2 (AA500-632), which are the two cystein-rich domains or in domain L2 (328-499), which is flanked by the two cystein-rich domains. The epitopes may also be located in any combination of domains such as, but not limited to, an epitope comprised by parts of L1 and S2.

The anti-HER-3 antibody used may be coupled to an effector and/or a labelling group. Corresponding coupling and/or labelling techniques are known to the person skilled in the art. Such effector and/or labelling groups may be attached for drug targeting, imaging applications and/or diagnostic applications.

As used herein, the term “labelling group” refers to a detectable marker, e.g. a radiolabelled amino acid or biotinyl moiety that can be detected by marked avidin (e.g. streptavidin bound to a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g. FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In certain aspects, it may be desirable that the labelling groups are attached by spacer arms of various lengths to reduce potential steric hindrance.

Alternatively, an anti-HER-3 antibody used according to the invention may be coupled to an effector group. As used herein, the term “effector group” refers to a cytotoxic group such as a radioisotope or radionuclide, a toxin, a therapeutic group or other effector group known in the art. Examples for suitable effector groups are radioisotopes or radionuclides (e.g. ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), calicheamicin, dolastatin analogs such as auristatins, and chemotherapeutic agents such as geldanamycin and maytansine derivates, including DM1. In certain aspects, it may be desirable that the effector groups are attached by spacer arms of various lengths to reduce potential steric hindrance.

In addition to the inhibition of HER-3, it is possible according to a further preferred embodiment to carry out or support the radiation treatment by labelling the anti-HER-3 antibody with a corresponding effector group such as radioisotopes or radionuclides outlined above.

Moreover, the HER-3 inhibitor used, and in particular the anti-HER-3 antibody used, according to the invention may be further characterized in that its binding to HER-3 reduces HER-3-mediated signal transduction. In accordance with the present invention, a reduction of HER-3-mediated signal transduction may, e.g. be caused by a downregulation of HER-3 resulting in an at least partial disappearance of HER-3 molecules from the cell surface or by a stabilization of HER-3 on the cell surface in a substantially inactive form, i.e. a form which exhibits a lower signal transduction compared to the non-stabilized form. Alternatively, a reduction of HER-3-mediated signal transduction may also be caused by influencing, e.g. decreasing or inhibiting, the binding of a ligand or another member of the HER family to HER-3, of GRB2 to HER-2 or of GRB2 to SHC, by inhibiting receptor tyrosine phosphorylation, AKT phosphorylation, PYK2 tyrosine phosphorylation or ERK2 phosphorylation, or by decreasing tumour invasiveness. Alternatively, a reduction of HER-3 mediated signal transduction may also be caused by influencing, e.g., decreasing or inhibiting, the formation of HER-3 containing dimers with other HER family members. One example among others may be the decreasing or inhibiting of the HER3-EGFR protein complex formation.

Preferred anti-HER-3 antibodies used according to the present invention are described in PCT/EP2006/012632.

According to an especially preferred embodiment the antibody comprises a heavy chain amino acid sequence that comprises at least one of the CDR's selected from the group consisting of (a) CDRH1's as shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230; (b) CDRH2's as shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230; and (c) CDRH3's as shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230

or a heavy chain amino acid sequence that shows at least 90%, 92%, 94%, 96%, 97%, 98%, 99% homology to one of the CDRs according to (a) to (c).

According to a further preferred embodiment the antibody comprises wherein the antibody comprises a light chain amino acid sequence that comprises at least one of the CDR's selected from the group consisting of: (d) CDRL1's as shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232; (e) CDRL2's as shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232; and (f) CDRL3's as shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232 or or a light chain amino acid sequence that shows at least 90%, 92%, 94%, 96%, 97%, 98%, 99% homology to one of the CDRs according to (d) to (f).

In yet another embodiment of the present invention the antibody comprises a heavy chain amino acid sequence that comprises at least one of the CDR's selected from the group consisting of

-   -   (a) CDRH1's as shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26,         30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84,         88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134,         138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186,         190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230;     -   (b) CDRH2's as shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26,         30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84,         88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134,         138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186,         190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230; and     -   (c) CDRH3's as shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26,         30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84,         88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134,         138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186,         190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230,

and a light chain amino acid sequence that comprises at least one of the CDR's selected from the group consisting of:

-   -   (d) CDRL1's as shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28,         32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98,         102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152,         156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204,         208, 212, 216, 220, 224, 228 and 232;     -   (e) CDRL2's as shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28,         32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98,         102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152,         156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204,         208, 212, 216, 220, 224, 228 and 232; and     -   (f) CDRL3's as shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28,         32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98,         102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152,         156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204,         208, 212, 216, 220, 224, 228 and 232.

According to a further preferred embodiment, the antibody comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3.

According to a further preferred embodiment, an anti-HER-3 antibody used according to the invention comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230.

A further preferred embodiment relates to the use of an anti-HER-3 antibody comprising a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232.

According to yet another preferred embodiment, an anti-HER-3 antibody used according to the invention comprises a heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230; and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232.

Especially preferred embodiments relate to anti-HER-3 antibodies comprising the heavy chain amino acid sequence of SEQ ID NO:42 and the light chain amino acid sequence of SEQ ID NO:44,

or comprising the heavy chain amino acid sequence of SEQ ID NO:54 and the light chain amino acid sequence of SEQ ID NO:56,

or comprising the heavy chain amino acid sequence of SEQ ID NO:70 and the light chain amino acid sequence of SEQ ID NO:72.

In particular, the anti-HER-3 antibody is selected from the group consisting of U1-1 antibody, U1-2 antibody, U1-3 antibody, U1-4 antibody, U1-5 antibody, U1-6 antibody, U1-7 antibody, U1-8 antibody, U1-9 antibody, U1-10 antibody, U1-11 antibody, U1-12 antibody, U1-13 antibody, U1-14 antibody, U1-15 antibody, U1-16 antibody, U1-17 antibody, U1-18 antibody, U1-19 antibody, U1-20 antibody, U1-21 antibody, U1-22 antibody, U1-23 antibody, U1-24 antibody, U1-25 antibody, U1-26 antibody, U1-27 antibody, U1-28 antibody, U1-29 antibody, U1-30 antibody, U1-31 antibody, U1-32 antibody, U1-33 antibody, U1-34 antibody, U1-35 antibody, U1-36 antibody, U1-37 antibody, U1-38 antibody, U1-39 antibody, U1-40 antibody, U1-41 antibody, U1-42 antibody, U1-43 antibody, U1-44 antibody, U1-45 antibody, U1-46 antibody, U1-47 antibody, U1-48 antibody, U1-49 antibody, U1-50 antibody, U1-51 antibody, U1-52 antibody, U1-53 antibody, U1-55.1 antibody, U1-55 antibody, U1-57.1 antibody, U1-57 antibody, U1-58 antibody, U1-59 antibody, U1-61.1 antibody, U1-61 antibody, U1-62 antibody or an antibody having at least one heavy or light chain of one of said antibodies. Especially preferred are the antibodies U1-49 (SEQ ID NO: 42/44), U1-53 (SEQ ID NO: 54/56) and U1-59 (SEQ ID NO: 70/72) or an antibody having at least one heavy or light chain of one of said antibodies. The antibody U1-59 is particular preferred.

The disease to be treated according to the present invention is a hyperproliferative disease. This hyperproliferative disease is preferably cancer, in particular squamous cell carcinoma.

Examples of cancer which are preferably treated according to the present invention are selected from the group consisting of breast cancer, gastrointestinal cancer, pancreas cancer, prostate cancer, ovarian cancer, stomach cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, testis cancer, soft tissue sarcoma, head and neck cancer, other HER-3 expressing or overexpressing cancers, and formation of tumour metastases.

According to an especially preferred embodiment the cancer is squamous cell carcinoma of the lung, of the head and/or the neck.

Radiation treatment according to the present invention is preferably selected from heat treatment, external beam radiation therapy, brachytherapy and/or radioisotopetherapy. The person skilled in the art can determine which kind of radiation treatment and in particular which kind of radiation source to be used for a particular patient to be treated.

External beam radiotherapy is the most common form of radiotherapy. In contrast to internal radiotherapy (brachytherapy), external beam radiotherapy directs the radiation at the tumour from outside the body. The voltage to be used may be determined by the person skilled in the art and depends inter alia on the kind of the tumour to be treated. For example, kilovoltage X-rays may be used for treating skin cancer and superficial structures whereas megavoltage X-rays may be used for treating deep-seated tumours (e.g., prostate, lung or brain). While X-ray and electron beams are by far the most widely used sources for external beam radiotherapy, also heavier particle beams such as proton beams may be used.

As already outlined above, brachytherapy is also known as internal radiotherapy. It is a form of radiotherapy, where a radiation source is placed inside or next to the area requiring treatment. In brachytherapy radiation sources are precisely placed directly at the side of the cancerous tumour. This means that the radiation only affects a limited local area which provides advantages over X-ray beam radiation therapy. For example, the tumour may be treated with very high doses of localized radiation whilst reducing the probability of unnecessary damage to surrounding healthy tissues. Examples of radiation sources used for brachytherapy comprise ¹²⁵I/¹⁰³Pd, ⁹⁰Y as well as Selective internal radiation therapy (SIRT). SIRT comprises the use of micospheres, which might be, for example, injected. Such microspheres can be made of resin or glass. Inside the microsphere the radiation source such as yttrium-90 can be placed. Examples of microspheres which are used already are ThereSphere and SIR-Spheres, which differ in their radioactivity per sphere and embolic effect. Of course, the use of any further suitable carrier coupled to a radiation source is also contemplated. Further examples for medicaments used in brachytherapy comprise SAVI, MammoSite, Contura, Proxcelan and I-Seed.

Radioisotope therapy represents a further form of targeted therapy. Targeting can be due to the chemical properties of the isotope. The radioisotopes may be, for example, delivered by infusion or ingestion. Examples of radioisotopes used for treatment comprise iodine-131, luteium-177 and yttrium-90, strontium-89 and samarium-153.

Radiation treatment and/or radiation treatment may be used according to the invention may be based on an single dose or fractionated dosing of radiation. Of course, is also possible to vary the duration of radiation therapy. The duration may depend on the location of the tumours to be treated, the size of the tumour to be treated and the physical condition of the patient.

The combination of an inhibitor of HER-3 and radiation therapy and/or treatment according to the present invention contemplates the simultaneous treatment with an HER-3 inhibitor and radiation therapy as well es a timely shifted combination. According to a preferred embodiment tumour cells to be treated are radiosensitized by the HER-3 inhibitor before radiation treatment, i.e. the HER-3 inhibitor is administered before radiation treatment/therapy.

The present invention can be used in combination with a further active compound like a further chemotherapeutic compound such as cytotoxic agents, such as doxorubicin, cis-platin or carboplatin, cytokines or antineoplatic agents.

Another aspect of the present invention relates to the use of an inhibitor of HER-3 for the modulation of radiosensitivity of cells.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1.

(A) HER3 profile in multiple carcinoma celllines.

Five human lung carcinoma celllines (NCI-H226, H292, H358, H520 and A549), five colorectal carcinoma celllines (Caco2; SW48, LS180, Lovo and HCT116) and five head and neck carcinoma celllines (SCC1, SCC6, SCC11A, SCC38 and SCC1483) were cultured in relevant mediums. Whole cell lysate was obtained with lysis buffer. Protein was quantitated using the Bradford method, sepreated by SDS gel and followed by immunoblot with the indicated antibodies.

(B) Basal activity of HER3 is blocked by U1-59.

Four head and neck carcinoma celllines (SCC6, SCC11A, SCC38, SCC1483) with high p-HER3 and two lung cancer cellines (H226 and A549) were treated with doses of U1-59 for 24 h. Whole cell lysates were isolated and followed by immumoblot with indicated antibodies. U1-59 inhibited HER3 activity and its downstream signal in a dose-dependent manner in those cells.

FIG. 2:

Radiation-induced activation of HER3 is blocked by U1-59. (A, D) HER3 was transient activated by radiation during the indicated time and blocked by U1-59. SCC6 and H226 cells were incubated with/without U1-59 (20 ug/nnl) for 24 h before radiation. After exposure to 4Gy radiation, whole cell lysates were isolated at the indicated times, followed by immunoblot with p-HER, p-AKT and p-MAPK antibodies. (B, E) U1-59 kept blocking activations of AKT and MAPK 24 h and 48 h after radiation. (C, F) Immunoflurescence showing HER3 were activated by radiation and blocked by U1-59 in SCC6 and H226 cells (30 minutes after radiation).

FIG. 3.

(A, F) U1-59 inhibited cell proliferation in a dose-dependent manner. SCC6 and H226 cells were seeded in 96-well plates and incubated with doses of U1-59 for 72 h. Cell proliferation were detected using CCK8 72 h. (B, G) Sensitizing effect of U1-59 on SCC6 cells in response to radiation. SCC6 and H226 were incubated with U1-59 for 4 h, and radiated with indicated doses. Clonegenic assay were performed as described. Control curves were exposed to radiation without U1-59 treatment.

(C, H) Impact of U1-59 on cell cycle.

SCC6 and H226 cells were incubated with U1-59 (20 ug/ml) for 48 h, followed by exposure to 6Gy radiation. 24 h after radiation, cell cycle (stained with PI) was detected using flow cytometer and analyzed by FlowJo. U1-59 caused G1 arrest and radiation cuased G2 arrest. Combination of U1-59 and radiation reassorted cell within cell cycle, induced accumulation of cells in G1 and G2, and reduced the population of cells in S phase.

(D, I) Impact of U1-59 on cell apoptosis.

Apoptosis (stained with annexin V/PI) was detected using flow cytometer and analyzed by FlowJo. Combination of U1-59 with radiation increased the percentage of apoptotic cells.

(E, J) U1-59 contribute to γ-H2AX focus formation.

SCC6 and H226 were incubated with U1-59 for 24 h before 4 Gy radiation. 4 h after radiation, cells were fixed and performed immunoflurescence staining. Compared with drug or radiation alone, increased number of γ-H2AX foci was detected in the cell treated with XRT combined with U1-59, demonstrated enhanced DNA damage by U1-59

FIG. 4.

Antitumour effect of U1-59 combined with radiation on xenograft tumours in athymic mice. SCC6, SCC1483 and H226 xenograft were performed as described in “Materials and methods”. (A) Mice were treated with single dose of IgG, radiation (10Gy or 16Gy), U1-59 (8 mg/kg) or combination of both. Data points were expressed as mean tumour size±SD. Results were graphed with tumour growth curve and Kaplan Meir survival curve. (B) Mice were treated with fractionated doses of IgG, radiation, U1-59 (100 ug/mouse) or combination of both twice a week as shown. Results were graphed with tumour growth curve and Kaplan Meir survival curve.

FIG. 5.

U1-59 inhibits basal and radiation-induced activation of HER3 in xenograft tumours. SCC6, SCC1483 and H226 xenograft tumours were collected at 24 h post radiation or the last radiation in fractionated treatment groups. Protein was isolated from the tumours and p-HER3 was analyzed by western blot (FIG. 5A and FIG. 5B). Increased p-HER3 was found in radiated tumours and reduced p-HER3 was found in tumours with U1-59 or combined treatment.

FIG. 6.

U1-59 inhibits cell survival signalsand enhances DNA damage in combination with radiation on xenograft tumours in athymic mice. SCC6 xenograft tumours with single dose treatment of radiation (16 Gy), U1-59 (8 mg/kg) or combination of both. tumours were harvested 24 h post treatment. IHC stain were performed as describe in “Materials and methods”. Reduced p-MAPK, p-S6 and PCNA staining, decreased PCNA positive cells and increased γ-H2AX positive cells were detected in tumours with U1-59 or combined treatment.

EXAMPLES

Materials and Methods

Cell Culture and Drug

Five human lung carcinoma celllines (NCI-H226, H292, H358, H520 and A549 and five colorectal carcinoma celllines (Caco2, SW48, LS180, Lovo and HCT116) were purchased from ATCC (Manassas, Va., USA) and maintained in 10% fetal bovine serum (FBS) in RPMl1640 or DMEM (Mediatech Inc., Manassas, Va., USA) with 1% penicillin and streptomycin. Five head and neck carcinoma celllines (UM-SCC1, UM-SCC4, UM-SCC6, UM-SCC11A, and UM-SCC1483 cells) were obtained from University of Michigan and maintained in 10% FBS (Invitrogen, Carlsbad, Calif., USA) in DMED supplemented with 1% hydrocortisone.

The anti-HER-3 antibody U1-59 was used.

Cell Proliferation Assay

Cells were seeded in 96-well plate and exposed to doses of U1-59 for 72 h. Cell proliferation was tested by Cell Counting Kit-8 (Dojindo Molecular Technologies, Gaithersbury, Md., USA).

Clonogenic Assay

A specified number of cells were seeded into each well of six-well tissue culture plates. After allowing cells time to attach (6 hours), U1-59 or the vehicle control (PBS) was added at specified concentrations. The plates were irradiated 4 hours later at the doses of 2, 4, 6, 8Gy. 10 to 14 days after seeding, colonies were stained with crystal violet, the number of colonies containing at least 50 cells was determined and the surviving fractions were calculated. Survival curves were generated after normalizing for cytotoxicity generated by U1-59 alone. Data presented are the mean±SD from at least three independent experiments.

Cell Cycle Analysis

Cells were incubated with U1-59 (20 ug/ml) for 48 h, followed 6Gy radiation. After 24 h, cells were trypsinized and washed with PBS, fixed in 90% ethanol and stored at 4° C. overnight. After remove of ethanol by centrifugation, cells were stained with PI stain buffer (50 ug/ml PI, 100 ug/ml Rnase A, 0.1% Triton X-100). Cells were sorted by FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA). Histogram analysis was performed with FlowJo software (Tree Star Inc., Ashland, Oreg., USA).

Apoptosis

Apoptosis were detected using Annexin V/PI kit. Following treatment, a cell suspension containing 1×10⁵ cells in 100 μl staining buffer was incubated with 5 μl Annexin V and PI. Cells were sorted by FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA). Population analysis was performed with FlowJo software.

Immunoblotting Analysis

Following treatment, cells were lysed with buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml of leupeptin and aprotinin). Protein was quantized using a standard Bradford absorbance assay. Equal amounts of protein were fractionated by SDS-PAGE. Thereafter, proteins were transferred to PVDF membrane and analyzed by incubation with the appropriate primary antibody. Proteins were detected via incubation with HRP-conjugated secondary antibodies and ECL chemiluminescence detection system. The NIH ImageJ program was used to measure densitometry of the western bands. The antibodies used in this study were as follows: HERS, AKT, MAPK, horseradish peroxidase-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif., USA). p-HER3 (Tyr1289), p-AKT and p-MAPK were obtained from Cell Signaling Technology (Beverly, Mass., USA). α-tubulin was from Calbiochem (San Diego, Calif., USA).

Immunofluorescence Assay

Approximately 2×10³ cells were seeded on a four-well glass chamber slide (Nalgene Nunc, Naperville, Ill., USA). Forty-eight hours later, cells were washed 3 times with PBS and fixed with 2% formaldehyde for 15 min at room temperature. Cells were incubated in ice-cold 100% methanol for 10 min at −20° C. and blocked with 5% normal serum in PBS with 0.3% Triton X100 solution for 1 h at room temperature and incubated with p-HER3 or λ-H2AX antibody overnight at 4° C. Next, cells were incubated with FITC-conjugated appropriate secondary antibody in PBS containing 0.3% Triton X100 and 1% BSA for 2 h. Slides were mounted using ProLong gold with DAPI (Invitrogen). Photographs were captured by confocal microscopy or fluorescence microscopy.

Mouse Xenograft Model

Athymic nude mice (4- to 6-week old; male) were obtained from the Harlan Laboratories (Indianapolis, Ind., USA). All animal procedures and maintenance were conducted in accordance with the institutional guidelines of the University of Wisconsin. Cells were injected bilaterally in the dorsal flanks of the mice at day 0 (2×106 cells). Once tumours reached expected volumes, Mice were single or fractionated dosed-treated with 1) IgG, 2) U1-59, 3) radiation or 4) the combination. Measurements were evaluated by digital calipers and calculated by the formula (π)/6×(large diameter)×(small diameter).

Immunohistochemistry

Xenograft Tumours were Fixed in Neutral Formalin and Embedded in Paraffin.

Immunohistochemical staining was performed for PCNA, p-MAPK, p-AKT, p-S6 and γ-H2AX. In brief, specimen was deparaffinized and rehydratded routinely, following antigene retrieval by citrate buffer for 15 mins at 98° C. in water bath, incubation in 3% hydrogen peroxide for 10 minutes, 3% BSA blocking for 30 min. Staining were performed as below: incubation in primary antibody diluted in recommended antibody diluents at 4° C. overnight, incubation in biotinylated secondary antibody for 30 minutes at room temperature, peroxidase visualization using Dakocytomation Liquid DAB+Substracte Chromogen System, Counterstain in Hematoxylin, routine Dehydrate and Clear, mounting with coverslip.

Statistical Analysis

Xenograft tumour growths were graphed and analyzed using GraphPad Prism 5 (GraphPad, San Diego, Calif.).

Results

HER3 is Expressed in Multiple Solid Tumour Cell Lines

Human epidermal growth factor receptor 3 (HER3) is a key dimerization partner for the HER family and activates oncogenic signaling pathways. Its overexpression in many solid tumours has been linked to poor prognosis. In FIG. 1A we charactered 15 cell lines derived from non-small cell lung carcinoma (H226, H292, H358, H520 and A549), colorectal carcinoma (Caco2, SW48, LS180, Lovo and HCT116) and head and neck squamous cell carcinoma (SCC1, SCC6, SCC11A, SCC38 and SCC1483) for the expression of both HER3 and p-HER3. Both HNSCC and NSCLC cell lines contain activated HER3 suggesting that HER3 may be enhancing their tumourigenic phenotype.

U1-59 can Inhibit Basal Activity of HER3 and Radiation-Induced Activation of HER3

HER3 lacks intrinsic tyrosine kinase activity. However, upon binding to multiple ligands, such as heregulin (neuregulin-1), HER3 can form heterodimers with other HER family member receptors to initiate the activation of multiple signaling pathways that strongly influence cell proliferation and survival. U1-59 is a fully humanized monoclonal antibody that binds to and inactivates HER3 oncogenic signaling pathways. In FIG. 1B, we demonstrate that treatment of multiple HNSCC and NSCLC cell lines with escalating doses of U1-59 can successfully inhibit the activation of both HER3 and AKT (FIG. 1B). In addition, using immunoblotting and immunofluorescence assays, we found that radiation treatment of the HNSCC cell line SCC6 and the NSCLC cell line H226 increased activated forms of HER3 (FIGS. 2A, 2C, 2D, and 2F). The transient activation of HER3 after radiation treatment was also accompanied by an increase in activation of both ERK and AKT in both SCC6 and H226 cell lines (FIGS. 2A and 2D). In FIGS. 2B and 2E we demonstrate that radiation can lead to the continual activation of HER3, AKT, and ERK in both SCC6 and H226 cell lines both 24 and 48 hours post radiation treatment. We subsequently demonstrate that the pre-incubation of either SCC6 or H226 cell lines with U1-59 for 24 hrs prior to radiation treatment could prevent the radiation-induced activation of HER3, ERK, and AKT (FIGS. 2A, 2C, 2D, and 2F). Additionally, U1-59 could lead to the continued inhibition HER3, ERK, and AKT activation at both 24 and 48 hours post radiation treatment (FIGS. 2B and 2E). Collectively, these data suggest that pre-incubation of cells with U1-59 prior to radiation treatment can prevent radiation induced activation of cell survival and proliferation pathways.

U1-59 can Radiosensitize HNSCC and NSCLC Cell Lines In-Vitro

In FIG. 2 we demonstrated that U1-59 could prevent radiation-induced activation of HER3 and subsequent downstream effectors ERK and AKT, therefore we hypothesized that U1-59 could enhance the radiosensitivity of both HNSCC and NSCLC cell lines. First, we demonstrate in FIGS. 3A and 3F that U1-59 can inhibit the growth of both SCC6 and H226 cell lines in a dose-dependent manner. To evaluate the in vitro effect of U1-59 combination with radiation, we conducted clonogenic assays as described in “materials and methods”. We pre-incubated both SCC6 and H226 cells with U1-59 for 4 hr, and subsequently subjected cells to radiation with the indicated doses. As shown in FIGS. 3B and 3G, U1-59 increased the slope of the radiation curves, demonstrating a sensitizing effect of U1-59 on both SCC6 and H226 cells in response to radiation. Control curves were exposed to radiation without U1-59 treatment.

U1-59 can Promote Cell Cycle Arrest and Apoptosis in Combination with Radiation Treatment In-Vitro

We further hypothesized that U1-59 may also enhance HNSCC and NSCLC cell lines to radiation induced cell cycle arrest and apoptosis. To study this we pre-incubated both SCC6 and H226 cells with U1-59 for 48 h followed by exposure to 6Gy radiation. 24 h post radiation we analyzed the cell cycle phase distribution and apoptotic levels by PI and annexin V staining. As shown in FIGS. 3C and 3H U1-59 pre-treated cells demonstrated a G1 phase arrest, while radiation treated cells demonstrated a G2 phase arrest as compared to control cells. We observe a decrease in S-phase with either treatment. It is well known that cells in G1 phase are more sensitive to radiation treatment, while S-phase cells are more resistant. Therefore we demonstrate through Annexin V staining that apoptosis is indeed enhanced in radiation treated cells pre-incubated with U1-59 (FIGS. 3D and 3I), suggesting that the G1 phase arrest induced by U1-59 may have promoted the radiosentitivy of both SCC6 and H226. In addition, we further show an enhanced level of DNA double strand breaks via γ-H2AX staining in SCC6 and H226 cells pre-treated with U1-59 prior to radiation (FIGS. 3E and 3J). Overall, these data demonstrate the enhanced antitumour effect of U1-59 in combination with radiation.

U1-59 in Combination with Radiation can have Antitumour Effects in HNSCC and NSCLC Mouse Tumour Xenografts

To further evaluate the efficacy of radiotherapy combined with U1-59, we inoculated SCC6, SCC1483 and H226 cell lines into athymic mice. Once tumours reached 100-200 mm³ tumours were divided up for both single and fractionated doses of radiation. In the single dose treated group, xenograft mice were administered with IgG, U1-59 (8 mg/kg), radiation (XRT16Gy or XRT10Gy) or a combination of both (FIG. 4A). In fractionated treated group, xenograft mice were administered with IgG, U1-59 (100 ug/mouse), radiation (twice a week at indicated doses), or combination (FIG. 4B). All three xenografts models demonstrated a marked reduction in tumour volume when treated with both U1-59 in combination with either a single dose or fractionated dose of radiation as compared to single therapy treated mice or IgG control treated mice. Additionally, xenografts treated with both U1-59 and either a single or fractionated dose of radiation demonstrate a clear survival advantage as depicted via Kaplan Meir survival curve analysis.

To validate that U1-59 could effectively inhibit the activation of HER3 in-vivo, we isolated protein from various tumours from each treatment group and performed western blot analysis for both total and activated forms of HER3.

In FIG. 5A we demonstrate that tumours isolated from mice treated with U1-59 alone or U1-59 in combination with a single dose of radiation have lower levels of HER3 activation. In SCC1483 and H226 xenograft models U1-59 treated tumours also contained lower total amounts of HER3 protein (FIG. 5A), something that was not observed in these cell lines treated with U1-59 in-vitro. Additionally, at least one of the two tumours isolated from each xenograft model in FIG. 5A demonstrated a clear activation of HER3 upon a single dose of radiation, which was blocked in the U1-59 treated tumours. In FIG. 5B we further demonstrate that U1-59 treated tumours in combination with fractionated doses of radiation also contained reduced levels of activated and total levels of HER3; activation of HER3 by fractionated doses of radiation was also blocked by U1-59 as well. Collectively, xenograft tumours treated with both U1-59 in combination with either a single dose or fractionated doses of radiation demonstrate a clear growth delay with marked reduction in tumour volumes as compared to controls or either treatment alone, suggesting that the loss of both activated and total levels of HER3 expression may radiosensitize HNSCC and NSCLC tumours.

U1-59 in Combination with Radiation can Prevent the Activation of Cell Survival Signals and Enhance DNA Damage In-Vivo

To further analyze the growth inhibitory effects of U1-59 in combination with radiation treatment, we analyzed the activation of ERK, AKT, S6 kinase, and γ-H2AX in SCC6 treated tumours via immunohistochemistry (FIG. 6). We demonstrate that SCC6 cells treated with both U1-59 and a single dose of radiation can have lower activation levels of the downstream effector molecules ERK, AKT, and S6 kinase. Additionally, U1-59 treated tumours demonstrated a decreased level of PCNA positive cells. Finally, increased levels of γ-H2AX were found in SCC6 tumours treated with both U1-59 and radiation. Overall, these data indicate that U1-59 in combination with radiation can efficiently inhibit cell survival and proliferation pathways, in addition to enhancing DNA damage. These data provide mechanistic evidence for the enhanced antitumour effect of U1-59 combined with radiation in vivo.

The results of these experiments indicate that the combination of U1-59 and radiation had a strong impact on tumour growth in studies using single dose or fractionated dosing of radiation. tumour analysis indicated that radiation treatment activated HER3 in vivo and U1-59 could abrogate this activation. Collectively our findings in vitro and in vivo indicate that U1-59 in combination with radiation has an impact on cell and tumour growth by delaying cell cycle progression, increasing apoptosis and increasing DNA damage. These findings indicate that HER3 may play an important role in response to radiation therapy and blocking its activity may be of strong therapeutic benefit in human tumours.

Table of CDR Sequences Ab Pat. chain ID: CDR1 CDR2 CDR3 heavy U1-1 GGSINSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 235) (SEQ ID NO: 236) (SEQ ID NO: 237) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 238) (SEQ ID NO: 239) (SEQ ID NO: 240) heavy U1-2 GGSISSGDYYWS YIYYSGSTYYNPSLRS ADYDFWSGYFDY (SEQ ID NO: 241) (SEQ ID NO: 242) (SEQ ID NO: 243) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 244) (SEQ ID NO: 245) (SEQ ID NO: 246) heavy U1-3 GGSISSGGYYWS YIYYSGSTYYNPSLKS DGYDSSGYYHGYFDY (SEQ ID NO: 247) (SEQ ID NO: 248) (SEQ ID NO: 249) light KSSQSVLYSSNNKNYLA WASTRES QQYYSTPLT (SEQ ID NO: 250) (SEQ ID NO: 251) (SEQ ID NO: 252) heavy U1-4 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 253) (SEQ ID NO: 254) (SEQ ID NO: 255) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 256) (SEQ ID NO: 257) (SEQ ID NO: 258) heavy U1-5 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 259) (SEQ ID NO: 260) (SEQ ID NO: 261) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 262) (SEQ ID NO: 263) (SEQ ID NO: 264) heavy U1-6 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWNGYFDY (SEQ ID NO: 265) (SEQ ID NO: 266) (SEQ ID NO: 267) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 268) (SEQ ID NO: 269) (SEQ ID NO: 270) heavy U1-7 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 271) (SEQ ID NO: 272) (SEQ ID NO: 273) light RASQDIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 274) (SEQ ID NO: 275) (SEQ ID NO: 276) heavy U1-8 GYTLTELSMY GFDPEDGETIYAQKFQG GWNYVFDY (SEQ ID NO: 277) (SEQ ID NO: 278) (SEQ ID NO: 279) light RSSQSLLHSNGYNYLD LDSHRAS MQALQTPLT (SEQ ID NO: 280) (SEQ ID NO: 281) (SEQ ID NO: 282) heavy U1-9 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWNGYFDY (SEQ ID NO: 283) (SEQ ID NO: 284) (SEQ ID NO: 285) light RASQDIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 286) (SEQ ID NO: 287) (SEQ ID NO: 288) heavy U1-10 GGSISSGDYYWS YIYYSGSTYYVPSLKS ADYDFWSGYFDY (SEQ ID NO: 289) (SEQ ID NO: 290) (SEQ ID NO: 291) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 292) (SEQ ID NO: 293) (SEQ ID NO: 294) heavy U1-11 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 295) (SEQ ID NO: 296) (SEQ ID NO: 297) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 298) (SEQ ID NO: 299) (SEQ ID NO: 300) heavy U1-12 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 301) (SEQ ID NO: 302) (SEQ ID NO: 303) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 304) (SEQ ID NO: 305) (SEQ ID NO: 306) heavy U1-13 GGSISSGGYYWS YIYYSGSTYYNPSLKS EDDGMDV (SEQ ID NO: 307) (SEQ ID NO: 308) (SEQ ID NO: 309) light RSSQSLLHSNGYNYLE LGSNRAS MQALQTPIT (SEQ ID NO: 310) (SEQ ID NO: 311) (SEQ ID NO: 312) heavy U1-14 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 313) (SEQ ID NO: 314) (SEQ ID NO: 315) light RASQGIRNDLG AASSLQS LQHNTYPWT (SEQ ID NO: 316) (SEQ ID NO: 317) (SEQ ID NO: 318) heavy U1-15 GGSVSSGGYYWS YIYYSGSTNYNPSLKS DGDVDTAMVDAFDI (SEQ ID NO: 319) (SEQ ID NO: 320) (SEQ ID NO: 321) light RASQSLSGNYLA GASSRAT QQYDRSPLT (SEQ ID NO: 322) (SEQ ID NO: 323) (SEQ ID NO: 324) heavy U1-16 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 325) (SEQ ID NO: 326) (SEQ ID NO: 327) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 328) (SEQ ID NO: 329) (SEQ ID NO: 330) heavy U1-17 GGSISSGDYYWS YIYYSGSTYYNSSLKS ADYDFWSGYFDY (SEQ ID NO: 331) (SEQ ID NO: 332) (SEQ ID NO: 333) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 334) (SEQ ID NO: 335) (SEQ ID NO: 336) heavy U1-18 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 337) (SEQ ID NO: 338) (SEQ ID NO: 339) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 340) (SEQ ID NO: 341) (SEQ ID NO: 342) heavy U1-19 GGSISSGDYYWS YIYYSGSTYYNPSLKS GDYDFWSGEFDY (SEQ ID NO: 343) (SEQ ID NO: 344) (SEQ ID NO: 345) light sequence not available heavy U1-20 GGSISSGGYYWS YIYDSGSTYYNPSLKS DQGQDGYSYGYGYYYGM (SEQ ID NO: 346) (SEQ ID NO: 347) DV (SEQ ID NO: 348) light QASQDISNYLN VASNLET QQCDNLPLT (SEQ ID NO: 349) (SEQ ID NO: 350) (SEQ ID NO: 351) heavy U1-21 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 352) (SEQ ID NO: 353) (SEQ ID NO: 354) light RASQDIRNDLG AASRLQS LQHNSYPWT (SEQ ID NO: 355) (SEQ ID NO: 356) (SEQ ID NO: 357) heavy U1-22 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 358) (SEQ ID NO: 359) (SEQ ID NO: 360) light RASQGIRNDLG AASSLQN LQHNSYPWT (SEQ ID NO: 361) (SEQ ID NO: 362) (SEQ ID NO: 363) heavy U1-23 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 364) (SEQ ID NO: 365) (SEQ ID NO: 366) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 367) (SEQ ID NO: 368) (SEQ ID NO: 369) heavy U1-24 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWNGYFDY (SEQ ID NO: 370) (SEQ ID NO: 371) (SEQ ID NO: 372) light RASQGIRNDLG AASSLQS LQHNNYPWT (SEQ ID NO: 373) (SEQ ID NO: 374) (SEQ ID NO: 375) heavy U1-25 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 376) (SEQ ID NO: 377) (SEQ ID NO: 378) light RASQGIRNDLG AASSLQN LQHNSYPWT (SEQ ID NO: 379) (SEQ ID NO: 380) (SEQ ID NO: 381) heavy U1-26 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDF (SEQ ID NO: 382) (SEQ ID NO: 383) (SEQ ID NO: 384) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 385) (SEQ ID NO: 386) (SEQ ID NO: 387) heavy U1-27 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDP (SEQ ID NO: 388) (SEQ ID NO: 389) (SEQ ID NO: 390) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 391) (SEQ ID NO: 392) (SEQ ID NO: 393) heavy U1-28 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGYFDS (SEQ ID NO: 394) (SEQ ID NO: 395) (SEQ ID NO: 396) light RASQGIRNDLG AASSLQS LQHNGYPWT (SEQ ID NO: 397) (SEQ ID NO: 398) (SEQ ID NO: 399) heavy U1-29 GFTFNSYDMH VIWYDGSNKYYADSVKG DRLCTNGVCYEDYGMDV (SEQ ID NO: 400) (SEQ ID NO: 401) (SEQ ID NO: 402) light QASQDISNYLN DASNLET QHYDTLPLT (SEQ ID NO: 403) (SEQ ID NO: 404) (SEQ ID NO: 405) heavy U1-30 GGSISSGDYYWS YIYYSGTTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 406) (SEQ ID NO: 407) (SEQ ID NO: 408) light RAGQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 409) (SEQ ID NO: 410) (SEQ ID NO: 411) heavy U1-31 GYTFTNYGIS WISAYDGYRNYAQKLQG DVQDYGDYDYFDY (SEQ ID NO: 412) (SEQ ID NO: 413) (SEQ ID NO: 414) light RASQSISSYLN AASSLQS QQSYSTPIT (SEQ ID NO: 415) (SEQ ID NO: 416) (SEQ ID NO: 417) heavy U1-32 GGSISSGDYYWS YIYYSGTTYYNPSLKS ADYDFWSGYFDY (SEQ ID NO: 418) (SEQ ID NO: 419) (SEQ ID NO: 420) light RAGQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 421) (SEQ ID NO: 422) (SEQ ID NO: 423) heavy U1-33 GGSISSGDYYWS YIYYSGSTYYNPSLKS ADYDFWSGHFDC (SEQ ID NO: 424) (SEQ ID NO: 425) (SEQ ID NO: 426) light RASQGIRDDLG AESSLQS LQHHSYPWT (SEQ ID NO: 427) (SEQ ID NO: 428) (SEQ ID NO: 429) heavy U1-34 GYTFTNYGIS WISAYDGYRNYAQKLQG DVQDYGDYDYFDY (SEQ ID NO: 430) (SEQ ID NO: 431) (SEQ ID NO: 432) light RASQSISSYLN AASSLQS QQSYSTPIT (SEQ ID NO: 433) (SEQ ID NO: 434) (SEQ ID NO: 435) heavy U1-35 GFTFSDYYMS YISSSGNNIYHADSVKG ERYSGYDDPDGFDI (SEQ ID NO: 436) (SEQ ID NO: 437) (SEQ ID NO: 438) light QASQDISNYLS DASNLET QQYDNPPCS (SEQ ID NO: 439) (SEQ ID NO: 440) (SEQ ID NO: 441) heavy U1-36 GGSISSGYYYWS YIYYSGTTYYNPSFKS ADYDFWSGHFDY (SEQ ID NO: 442) (SEQ ID NO: 443) (SEQ ID NO: 444) light RASQGIRNDLG AASSLQS LQHNSYPWT (SEQ ID NO: 445) (SEQ ID NO: 446) (SEQ ID NO: 447) heavy U1-37 GYTFTSYGIS WISAYDGHTNYAQKLQG DPHDYSNYEAFDF (SEQ ID NO: 448) (SEQ ID NO: 449) (SEQ ID NO: 450) light RASQSISSYLN AASSLQS QQSYSTPIT (SEQ ID NO: 451) (SEQ ID NO: 452) (SEQ ID NO: 453) heavy U1-38 GFSLSTSGVGVG LIYWNDDKRYSPSLKS RDEVRGFDY (SEQ ID NO: 454) (SEQ ID NO: 455) (SEQ ID NO: 456) light RSSQSLVYSDGYTYLH KVSNWDS MQGAHWPIT (SEQ ID NO: 457) (SEQ ID NO: 458) (SEQ ID NO: 459) heavy U1-39 GFTVSSNYMS VIYSGGSTYYADSVKG GQWLDV (SEQ ID NO: 460) (SEQ ID NO: 461) (SEQ ID NO: 462) light RSSQSLLHSNGYNYLD LGFHRAS RQALQTPLT (SEQ ID NO: 463) (SEQ ID NO: 464) (SEQ ID NO: 465) heavy U1-40 GGSISSGGYYWS YIYSSGSTYYNPSLKS DRELELYYYYYGMDV (SEQ ID NO: 466) (SEQ ID NO: 467) (SEQ ID NO: 468) light RSSQSLLYSNGYNYLD LGSNRAS MQALQTPLT (SEQ ID NO: 469) (SEQ ID NO: 470) (SEQ ID NO: 471) heavy U1-41 GGSISSGGYYWS YIYYSGSTYYNPSLKS DRELEGYSNYYGVDV (SEQ ID NO: 472) (SEQ ID NO: 473) (SEQ ID NO: 474) light RASQAISNYLN AASSLQS QQNNSLPIT (SEQ ID NO: 475) (SEQ ID NO: 476) (SEQ ID NO: 477) heavy U1-42 GYSFTSYWIG IIYPGDSDTRYSPSFQG HENYGDYNY (SEQ ID NO: 478) (SEQ ID NO: 479) (SEQ ID NO: 480) light RASQSIRSYLN AASSLQS QQSNGSPLT (SEQ ID NO: 481) (SEQ ID NO: 482) (SEQ ID NO: 483) heavy U1-43 GGSISSGGYYWS YIYYSGSTYYNPSLRS DREREWDDYGDPQGMDV (SEQ ID NO: 484) (SEQ ID NO: 485) (SEQ ID NO: 486) light RASQSISSYLH AASSLQS QQSYSNPLT (SEQ ID NO: 487) (SEQ ID NO: 488) (SEQ ID NO: 489) heavy U1-44 GYSFTSYWIG IIWPGDSDTIYSPSFQG HENYGDYNY (SEQ ID NO: 490) (SEQ ID NO: 491) (SEQ ID NO: 492) light RASQSIRSYLN AASSLQS QQSISSPLT (SEQ ID NO: 493) (SEQ ID NO: 494) (SEQ ID NO: 495) heavy U1-45 GYTFTSYDIN WMNPWSGDTGYAQVFQG FGDLPYDYSYYEWFDP (SEQ ID NO: 496) (SEQ ID NO: 497) (SEQ ID NO: 498) light RASQSISSYLN AASSLQS QQSYSTPLT (SEQ ID NO: 499) (SEQ ID NO: 500) (SEQ ID NO: 501) heavy U1-46 GDSVSSNSAAWN RTYYRSKWYNDYAVSVK DLYDFWSGYPYYYGMDV (SEQ ID NO: 502) S (SEQ ID NO: 504) (SEQ ID NO: 503) light sequence not available heavy U1-47 GDSVSSNSAAWN RTYYRSKWYNDYAVSVK DYYGSGSFYYYYGMDV (SEQ ID NO: 505) S (SEQ ID NO: 507) (SEQ ID NO: 506) light RASQSISSYLN AASNLQS QQSYSTPRT (SEQ ID NO: 508) (SEQ ID NO: 509) (SEQ ID NO: 510) heavy U1-48 GGSISSYYWS HIYTSGSTNYNPSLKS EAIFGVGPYYYYGMDV (SEQ ID NO: 511) (SEQ ID NO: 512) (SEQ ID NO: 513) light sequence not available heavy U1-49 GYTFTGYYMH WINPNIGGTNCAQKFQG GGRYSSSWSYYYYGMDV (SEQ ID NO: 514) (SEQ ID NO: 515) (SEQ ID NO: 516) light KSSQSLLLSDGGTYLY EVSNRFS MQSMQLPIT (SEQ ID NO: 517) (SEQ ID NO: 518) (SEQ ID NO: 519) heavy U1-50 GGSVSSGGYYWS YIYYSGSTNYNPSLKS GGDSNYEDYYYYYGMDV (SEQ ID NO: 520) (SEQ ID NO: 521) (SEQ ID NO: 522) light RASQSISIYLH AASSLQS QQSYTSPIT (SEQ ID NO: 523) (SEQ ID NO: 524) (SEQ ID NO: 525) heavy U1-51 GGSISSYYWS YIYYSGSTYYNPSLKS DSSYYDSSGYYLYYYAM (SEQ ID NO: 526) (SEQ ID NO: 527) DV (SEQ ID NO: 528) light KSSQSVLYSSNNKNYLA WASTRES QQYYTTPLT (SEQ ID NO: 529) (SEQ ID NO: 530) (SEQ ID NO: 531) heavy U1-52 GGSISSGGYYWS NIYYSGSTYYNPSLKS GGTGTNYYYYYGMDV (SEQ ID NO: 532) (SEQ ID NO: 533) (SEQ ID NO: 534) light RASQSVSSSYLA GASSWAT QQYGSSPLT (SEQ ID NO: 535) (SEQ ID NO: 536) (SEQ ID NO: 537) heavy U1-53 GFTFSIYSMN YISSSSSTIYYADSVKG DRGDFDAFDI (SEQ ID NO: 538) (SEQ ID NO: 539) (SEQ ID NO: 540) light QASQDITNYLN DASNLET QQCENFPIT (SEQ ID NO: 541) (SEQ ID NO: 542) (SEQ ID NO: 543) heavy U1-55.1 GGSVSSGGYYWN YINYSGSTNYNPSLKS DRELELYYYYYGMDV (SEQ ID NO: 544) (SEQ ID NO: 545) (SEQ ID NO: 546) light will be same as U1-55 heavy U1-55 will be same as U1-55.1 light RSSQSLLYSNGYKYLD LGSNRAS MQALQTPIT (SEQ ID NO: 547) (SEQ ID NO: 548) (SEQ ID NO: 549) heavy U1-57.1 will be same as U1-57 light RSSQSLLYSNGYKYLD LGSNRAS MQALQTPIT (SEQ ID NO: 550) (SEQ ID NO: 551) (SEQ ID NO: 552) heavy U1-57 GGSVSSGGYYWN YINYSGSTNYNPSLKS DRELELYYYYYGMDV (SEQ ID NO: 553) (SEQ ID NO: 554) (SEQ ID NO: 555) light will be same as U1-57.1 heavy U1-58 GFTFSSYGMH VIWYDGSNKYYADSVKG AARLDYYYGMDV (SEQ ID NO: 556) (SEQ ID NO: 557) (SEQ ID NO: 558) light RASQSINSYLN GASGLQS QQSYSSPLT (SEQ ID NO: 559) (SEQ ID NO: 560) (SEQ ID NO: 561) heavy U1-59 GGSFSGYYWS EINHSGSTNYNPSLKS DKWTWYFDL (SEQ ID NO: 562) (SEQ ID NO: 563) (SEQ ID NO: 564) light RSSQSVLYSSSNRNYLA WASTRES QQYYSTPRT (SEQ ID NO: 565) (SEQ ID NO: 566) (SEQ ID NO: 567) heavy U1-61.1 GVSISSGGYYWS YIYYSGSTYYNPSLKS DSESEYSSSSNYGMDV (SEQ ID NO: 568) (SEQ ID NO: 569) (SEQ ID NO: 570) light RASQTISSYLN AASSLQG QQSYSNPLT (SEQ ID NO: 571) (SEQ ID NO: 572) (SEQ ID NO: 573) heavy U1-61 GVSISSGGYYWS YIYYSGSTYYNPSLKS DSESEYSSSSNYGMDV (SEQ ID NO: 574) (SEQ ID NO: 575) (SEQ ID NO: 576) light will be the same U1-61.1 heavy U1-62 GYSFTSYWIG IIYPGDSDTRYSPSFQG QMAGNYYYGMDV (SEQ ID NO: 577) (SEQ ID NO: 578) (SEQ ID NO: 579) light RASQSVISIYLA GASSRAT QQYGSSPCS (SEQ ID NO: 580) (SEQ ID NO: 581) (SEQ ID NO: 582) 

We claim:
 1. A method of treating lung cancer in a subject comprising: administering to the subject an anti-HER-3 antibody or an antigen-binding fragment thereof in combination with radiation treatment, wherein the radiation treatment comprises X-ray radiation, wherein the anti-HER-3 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence comprising SEQ ID NO:70 and a light chain variable region with an amino acid sequence comprising SEQ ID NO:72, and wherein the anti-HER-3 antibody or antigen-binding fragment thereof is administered before the X-ray radiation and sensitizes the lung cancer to the X-ray radiation.
 2. The method of claim 1, wherein the anti-HER-3 antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a multispecific antibody, or an antigen-binding fragment thereof.
 3. The method of claim 1, wherein said antigen-binding fragment of the anti-HER-3 antibody is a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fv fragment, a diabody, or a single chain antibody molecule.
 4. The method of claim 1, wherein said anti-HER-3 antibody is coupled to an effector group and/or labelling group.
 5. The method of claim 4, wherein said effector group is a radioisotope, a radionuclide, a toxin, a therapeutic group, and/or a chemotherapeutic group.
 6. The method of claim 4, wherein said labelling group is a radioisotope or radionuclide, a fluorescent group, an enzymatic group, a chemiluminescent group, a biotinyl group, or a predetermined polypeptide epitope.
 7. The method of claim 1, wherein the lung cancer is squamous cell carcinoma of the lung.
 8. The method of claim 1, wherein the radiation treatment further comprises brachytherapy, radioisotope therapy, or heat treatment.
 9. The method of claim 1, wherein the radiation treatment is based on a single dose or fractioned dosing of radiation.
 10. The method of claim 1, wherein the method comprises administering a further active compound.
 11. The method of claim 10, wherein the further active compound comprises a chemotherapeutic compound.
 12. The method of claim 1 wherein the anti-HER-3 antibody or antigen-binding fragment thereof is administered 4 hours or 48 hours before the X-ray radiation.
 13. The method of claim 12 wherein the X-ray radiation is 10 Gy or 16 Gy.
 14. The method of claim 13 wherein the X-ray radiation is fractionated.
 15. The method of claim 13 wherein the X-ray radiation is a single dose.
 16. The method of claim 1 wherein the lung cancer is non-small cell lung cancer.
 17. A method of treating lung cancer in a subject, the method comprising: administering to the subject an anti-HER-3 antibody or an antigen-binding fragment thereof, wherein the anti-HER-3 antibody or an antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence comprising SEQ ID NO:70 and a light chain variable region with an amino acid sequence comprising SEQ ID NO:72, and subsequently administering to the subject a radiation treatment, the radiation treatment comprising X-ray radiation, wherein the anti-HER-3 antibody or antigen-binding fragment thereof sensitizes the lung cancer to the X-ray radiation.
 18. The method of claim 17 wherein the anti-HER3 antibody or antigen-binding fragment thereof is administered to the subject 4 hours or 48 hours prior to the X-ray radiation.
 19. The method of claim 18 wherein the X-ray radiation is 10 Gy or 16 Gy.
 20. The method of claim 19 wherein the X-ray radiation is fractionated.
 21. The method of claim 19 wherein the X-ray radiation is a single dose.
 22. The method of claim 17 wherein the lung cancer is squamous cell carcinoma of the lung.
 23. The method of claim 17 wherein the lung cancer is non-small cell lung cancer. 